Tissue-engineered ligament

ABSTRACT

An apparatus and method for the reconstruction of a previously torn ligament using a tissue-engineered ligament. The tissue-engineered ligament includes a scaffold of biocompatible material having at least one layer and forming a sheet. The scaffold is placed in a cultured medium for seeding with fibrocyte forming cells. The seeded scaffold is then placed in an incubator to increase the number of cells. The seeded scaffold is then formed into a slender structure suitable for implantation. The method of making a tissue-engineered ligament includes forming a scaffold of biocompatible material having at least one layer forming a sheet. Next, the scaffold sheet is seeded with fibrocyte forming cells. The method further includes increasing the number of cells on the seeded scaffold and forming a slender structure suitable for implantation from the scaffold.

REFERENCE TO EARLIER APPLICATION

[0001] This application claims the benefit of pending U.S. ProvisionalPatent Application Serial No. 60/165,331, filed Nov. 12, 1999 by JamesCho Hong Goh and Kwan-Ho Chan. The aforementioned document is herebyincorporated herein by reference.

FIELD OF THE INVENTION

[0002] This invention generally relates to apparatus and methods for themanufacture of replacement tissue using tissue engineering methods. Moreparticularly, this invention relates to apparatus and methods for themanufacture of tissue-engineered ligaments suitable for the treatment ofligament deficiencies in patients.

BACKGROUND OF THE INVENTION

[0003] Men and women who are athletically active experience the majorityof ligament tears, particularly tearing of the anterior cruciateligament of the knee. The anterior cruciate ligament is commonly torn byforces applied to the knee during twisting, cutting, deceleration ortackling. A torn anterior cruciate ligament will generally not heal. Ananterior cruciate deficient knee is often unstable during pivotingactivity. Repeated instability episodes of the knee may lead to furtherdamage of the articular surface and cause tearing in the menisci. It istherefore desirable to stabilize the knee by reconstructing a tornanterior cruciate ligament. Attempts in the past to directly repair thetorn anterior cruciate ligaments have been relatively ineffective.Prosthetic ligament replacements made of carbon fibers and Gore-Texmaterials do not last a long period of time. Repeated loading of aprosthetic ligament in a young active patient leads to failure of theligament. The release of debris from a failed ligament results inchronic inflammation of the joint, and osteolysis of bone, in and aroundthe area of ligament attachments.

[0004] The current standard practice is to reconstruct a torn anteriorcruciate ligament by substituting the torn ligament with a patient's owntissue. The middle third of the patellar tendon or the hamstring tendonsare commonly used as substitution ligaments. Alternatively, theallograft patellar tendon, hamstring tendon or Achilles tendon from adonor can be used for reconstructing the ligament. However, donormaterials are associated with a risk of infectious disease transmissionsuch as AIDS. Using a patient's own tissue is also associated withmorbidity at the donor site. For example, stress fracture of thepatellar, quadriceps muscle weakness and a long rehabilitation periodmay result from the use of a patient's own tissue. Furthermore,harvesting and preparation of autogeneous tissue prolongs surgery time.

[0005] Previous attempts to use an artificial stent to replace a damagedanterior cruciate ligament have not been successful. One such example isthe LAD Prosthetic Ligament, which was used as a scaffold for tissueingrowth. The LAD Prosthetic Ligament is not bioabsorbable. Therefore,whatever initial fibrous tissue that forms on the LAD ProstheticLigament is not subject to accommodating increasing loads and there isno stimulus for the fibrous tissue to proliferate to support increasingloads. Furthermore, the LAD Prosthetic Ligament is not an optimalstructure for tissue ingrowth.

[0006] Recent progress in tissue engineering has made it possible toharvest cells from a patient's own body or a donor. The harvested cellsare then grown into the desired tissues on three-dimensional scaffolds,or hydrogel carriers, made of biodegradable polymers. These tissuesinclude, but are not limited to, heart muscles, fat, cartilage, andskin. The tissue grown outside of the body, together with the scaffoldcontaining the tissue, is then transplanted into the patient to correctan existing defect. After transplantation, the cells may furtherreplicate, reorganize and mature, depending on the environment of thehost bed into which the cells were transplanted.

[0007] Two good sources of cells that are suitable for tissueengineering are embryonic stem cells and mesenchymal stem cells. Thesestem cells, when exposed to particular bioactive factors, also known asgrowth factors, can be directed to differentiate into different types ofcell lines in a predictable way. For example, mesenchymal stem cells canbe directed to differentiate into different types of tissue such as, butnot limited to, skin, tendon, ligament and bone under suitableconditions. These conditions include exposing cells to certain growthfactors. It is known that mesenchymal cells are directed todifferentiate into fibroblast when exposed to interleukin. Furthermore,fibroblast is only able to differentiate into fibrocytes that are themature cells of ligament tissue.

[0008] Mesenchymal cells are present in very small numbers in bonemarrow, periosteum, skin and muscle. A small piece of the tissuecontaining a small number of mesenchymal cells is preferably harvestedfrom the patient's own body. For example, a piece of periosteal tissueharvested from the patient or donor is morsellised into small pieces.Using tissue culture techniques well known to those skilled in the art,the mesenchymal cells are isolated and the number of cells expanded. Themesenchymal cells are then seeded onto scaffolds. These scaffolds arepreferably made of biodegradable materials to make the desired tissues.

[0009] There are two major challenges in growing tissue-engineeredligaments outside the body. First, most cells cultured in vitro tend togrow in a monolayer. Even if it is possible to culture tissue to a fewmillimeters thick, deeper layers of the cells may not have sufficientsupplies of nutrients. Secondly, it is difficult to adequately anduniformly seed the scaffold with cells to initiate cell expansion.

[0010] It is, therefore, an object of the present invention to providetissue-engineered ligaments for reconstruction of previously tornligaments.

[0011] It is another object of the present invention to providetissue-engineered ligaments to reduce the time it takes to complete thesurgery and to eliminate donor site morbidity in the patient.

[0012] It is still another object of the present invention to providetissue-engineered ligaments grown from a small amount of tissue obtainedfrom the patient.

[0013] It is another object of the present invention to provide ascaffold for uniform and adequate seeding of cells to initiate cellexpansion for making tissue-engineered ligaments.

[0014] It is also another object of the present invention to provide ascaffold for a tissue-engineered ligament with adequate channels fornutrients to reach the cells.

[0015] It is also another object of the present invention to provide amethod to enhance the growth and alignment of the fibrocytes and theextra cellular matrix during incubation of the tissue-engineeredligament.

[0016] Another object of the present invention is to providetissue-engineered ligaments that will permanently anchor to a patient'sbone.

[0017] Yet another object of the present invention is to providetissue-engineered ligaments that will mature and resist physiologicalload across the joint.

[0018] Still another object of the present invention is to provide amethod of making tissue-engineered ligaments.

SUMMARY OF THE INVENTION

[0019] The present invention comprises an apparatus and method for thereconstruction of a previously torn ligament using a tissue-engineeredligament. The tissue-engineered ligament includes a scaffold ofbiocompatible material having at least one layer and forming a sheet.The scaffold is placed in a cultured medium for seeding with fibrocyteforming cells. The seeded scaffold is then placed in an incubator toincrease the number of cells. The seeded scaffold is then formed into aslender structure suitable for implantation. The method of making atissue-engineered ligament includes forming a scaffold of biocompatiblematerial having at least one layer forming a sheet. Next, the scaffoldsheet is seeded with fibrocyte forming cells. The method furtherincludes increasing the number of cells on the seeded scaffold andforming a slender structure suitable for implantation from the scaffold.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] Referring first to FIGS. 1-12, the starting material for atissue-engineered ligament 10 is a sheet 15 of biocompatible materialwhich is preferably bioabsorbable. The sheet 15 may also be a porousstructure. The pores of the porous structure may be partially or fullyinterconnected across the thickness of the sheet. The pore structureessentially forms perforations when fully and directly interconnectedacross the thickness of the sheet. A sheet of woven fabric made of abiocompatible and preferably biodegradable material is an example ofsuch a construct. Additionally, the sheet may contain slits 20 whichextend completely through sheet 15. A method of forming atissue-engineered ligament includes placing the biocompatible sheet 15in a cultured medium and seeding the sheet with fibrocyte forming cells25 (FIG. 2). Next, the seeded sheet is incubated to increase the numberof fibrocyte forming cells. After a sufficient cell increase, sheet 15is reformed into a slender structure 35, such as a tissue-engineeredligament suitable for implantation into a patient. In this respect itshould be appreciated that the presence of slits 20 in sheet 15 permitnutrients and the like to pass readily into the interior of slenderstructure 35. In addition, slits 20 permit cells 25 to grow throughsheet 15. Furthermore, the presence of slits 20 provides greaterflexibility to slender structure 35, whereby it may function more like anatural ligament. If there is sufficient porosity in biocompatible sheet15, additional perforations or slits may not be necessary for diffusionof nutrients to cells 25 when sheet 15 is formed into slender structure35.

[0021] The first embodiment of the invention shown in FIGS. 1-4 providesrectangular sheet 15 of biocompatible material with multiplelongitudinal slits 20 precut into the material. Slits 20 start and endat a prescribed distance from the edge of rectangular sheet 15, therebyleaving uncut margins 30 connecting the adjacent strips as shown in FIG.2. The whole construct is immersed in culture medium solution (notshown) and then seeded with fibrocyte forming cells such as mesenchymalstem cells or fibroblast cells. When a sufficient quantity of fibroustissue has formed, sheet 15 is rolled into a slender structure 35,preferably with the strips oriented longitudinally and the uncut edges30 forming the ends of the slender structure. Slender structure 35 isthen implanted into a patient to reconstruct a missing ligament. Slenderstructure 35 may be further incubated prior to implantation to allow forfurther growth of fibrous tissue. Alternatively, slender structure 35can be implanted in a penultimate location in a patient's body such asthe peritoneal cavity, muscle bed or any other soft tissue bed. Thisimplantation allows further growth of fibrous tissue prior toimplantation in a functional location, such as the knee joint forreconstruction of the anterior cruciate ligament.

[0022] Other methods of forming slender structure 35 from sheet 15 arealso possible. Examples include, but are not limited to, folding sheet15 back and forth in an accordion style fold 40 with a width 45corresponding to the desired width of tissue-engineered ligament 10 asshown in FIGS. 5-8, or cutting sheet 15 into strips 50 corresponding toa desired width 55 of tissue-engineered ligament 10 and stacking onestrip 50 on top of another, thereby forming stack 60 as shown in FIGS.9-12.

[0023] The fibrocyte forming cells can be harvested from a human donor.Preferably, fibrocyte forming cells are harvested from the patient's ownbody. Fibrocyte forming cells, which include mesenchymal stem cells orfibroblast cells, can be derived from a number of sources such as skin,bone marrow and periosteum.

[0024] The biocompatible material of sheet 15, which is preferably alsoa bioabsorbable material, can be, but is not limited to, one or more ofthe following: polyglycolic acid, polylactic acid, a mixture of PGA/PLA,chitin and collagen. This material may also be porous. Sheet 15 maycomprise a uniform structure, a woven structure, a composite structure(e.g., a sheet with incorporated filaments, including aligned filamentssuch as for reinforcement), etc. Flat sheets 15 of autograft orallograft tissue may also be used. One example is fascia lata. Thefascia lata tissue can be preprocessed to reduce reaction. Severalmethods, including freeze-drying, exist for preprocessing fascia lata.

[0025] The biocompatible material of sheet 15 may also be coated withcollagen and other factors to promote adhesion of the fibrocyte formingcells 25. The expansion of the number of cells can be promoted by theaddition of growth factors to the biocompatible material of sheet 15 orthe culture medium.

[0026] To induce mesenchymal cells to differentiate into fibroblast,fibroblast growth factors such as interleukin may be added to thebiocompatible material or the culture medium.

[0027] The strength of the cultured fibrous tissue is made stronger byorientating the growth of the fibrocytes and deposition of the collagenfibers in the longitudinal direction of the slender structure. Thefibrocytes are induced to orientate longitudinally by incorporatinglongitudinal microchannels 201 (FIG. 2A), with a width and depth of theorder 1 to 200 microns, on the surface of the biocompatible material ofsheet 15. The longitudinal microchannels 201 encourage the fibrocytes tocluster along the microchannels and, additionally, urges the fibrocytecells to orient themselves parallel to the axis of the microchannels.The application of cyclic loading to the biocompatible materials duringincubation further enhances orientation of the cells and collagenfibers. Cyclic loading can also increase the growth of the fibroustissue. The cyclic loading can be applied to the biocompatible materialin the rolled or unrolled configuration. It is to be noted that when theslender structure is loaded in longitudinal tension, the strips 105 willbe taut and fluid will be forced out through the perforations or slits.When the tension is released or lessened, the slender structure tends toassume a slightly larger diameter, thus permitting fluid to flow intothe slender structure through the slits or perforations. Such cyclicloading will create an environment of circulating fluid providing freshnutrient fluid to the cells within the slender structure. Additionally,the cells suspended in the fluid for seeding are carried by thecirculating fluid into the interior of the slender structure 35 therebyincreasing the chance of cells attaching in the interior of the slenderstructure 35.

[0028] In an alternative embodiment of this invention (not shown),fibrocyte forming cells 25 on the biocompatible material of sheet 15 arecultured and, when a sufficient expansion of cells 25 is achieved, slits20 are then made in the sheets as described above. Some of cells 25 willbe damaged during the creation of slits 20. This damage is compensatedfor by the more efficient cell expansion on an uninterrupted (i.e.,slit-less) flat surface during the incubation period.

[0029] In order to increase the rate at which the number of cells 25increase, the mesenchymal stem cells or fibroblast can be geneticallyaltered to increase local production of desired growth factors. This canbe done by viral transfection or by incorporating plasmid genes into thematrix of the biocompatible material.

[0030] Now looking at FIG. 13, the ligament 10 is ready fortransplantation into a patient after incubating cells 25 attached toscaffold sheet 15. Incubation usually occurs for a period of timeranging from one to twelve weeks to allow substantial growth andexpansion of cells 25. In general, as shown in FIG. 13, two bone tunnels65 are prepared during surgery on opposite sides of the joint usingtechniques well known in the art. The ligament 10 is then fixed in thebone tunnels 65 with an interference screw 70 at both ends. This issimilar to the fixation of hamstring ligaments with an interferencescrew in standard arthroscopic anterior cruciate reconstruction.Alternatively, if desired, other surgical techniques well known in theart may be used to secure the tissue-engineered ligament 10 within theknee joint. Without harvesting autologous tissue, such as the patellartendon or hamstring tendons, the complexity and time required forcompleting this surgery is greatly reduced. Also any morbidityassociated with the harvesting of autologous tendon is eliminated.

[0031] After successful implantation of tissue-engineered ligament 10,further growth of the fibroblast and fibrocytes will further anchorligament 10 in the bone tunnels 65 by tissue ingrowth. Also repeatedcyclic loading will encourage hypertrophy of tissue-engineering ligament10. With time, the scaffold sheet 15 will be gradually absorbed and theentire load across the joint will eventually be carried bytissue-engineered ligament 10. Ligament 10 is a live tissue and willeventually mature into a more stable structure capable of resistingnormal transient increases in physiologic load.

[0032] Now looking at FIGS. 14-16, fixation can be enhanced by moldingthreads 75 into the ends 80 of slender structure 35 using a moldingdevice 85 before implantation. Threaded ends 75 of tissue-engineeredligament 10 engage the thread of interference screw 70, as seen in FIG.17. Advancement of interference screw 70 urges threaded end 75 oftissue-engineered ligament 10 against the wall of bone tunnel 65 andcauses threads 75 of end 80 of tissue-engineered ligament 10 to embedinto bone tunnel 65. This action further enhances fixation.Alternatively, a rigid body (not shown) secured to the end of thetissue-engineered ligament may be provided for an interference screw topress against to provide fixation. The rigid body may contain threadscorresponding to the screw. Alternatively, the rigid body may not haveany threads.

[0033] In a preferred embodiment, the starting material is a thinrectangular sheet 15 of a biocompatible material including abiodegradable polymeric material, a natural material, and/or anallograft or autograft fascia lata material. Examples of biodegradablepolymeric materials include, but are not limited to, PGA, PLA, ormixture of PLA/PGA, etc. Examples of natural materials include, but arenot limited to chitin, chitosan, etc.

[0034] Looking at FIG. 18, prescribed sections 85 at both ends of sheet15 may be thicker than the rest of sheet 15. In a preferred embodiment,sections 85 have a specific male pattern 90 on one side and matchingfemale pattern 95 on the other. The matching male and female patterns90, 95 contact and inter-digitate when sheet 15 is rolled up. Theseinter-digitations at both ends of the sheet prevent sliding betweenadjacent layers of the rolled-up structure to maintain a stable, slenderstructure 35. Also, the slightly greater thickness at both ends helps toseparate the layers to provide room for cellular growth between thelayers.

[0035] In this configuration, a middle thinner portion 100 of sheet 10has many tiny strips 105. These strips 105 are made with multiplelongitudinal slits pre-cut into the material. The slits start and end ata prescribed distance from the edge of the rectangular material to leaveuncut margins 30 connecting the adjacent strips 105. These uncut margins30 form the earlier mentioned thicker sections preferably havinginterlocking patterns. Each of the tiny strips 105 has longitudinalmicrochannels 201. These microchannels each have a width and depth ofthe order of 1 to 200 microns. The sheet may be hydrophilised and coatedwith collagen and growth factors such as TGF beta, IGF, etc.

[0036] Cell seeding may be performed using various methods consistentwith the present invention. Several such methods will now be set forthby way of example but not limitation.

EXAMPLE I

[0037] One method involves clamping sheet 15 at both ends 80 of thethicker, uncut sections and immersing the sheet in a culture medium 110.Sheet 15 is initially held in tension and mesenchymal stem cells orfibroblasts are introduced into culture medium 110 and onto strips 105.Cells 25 are allowed to attach to strips 105 and proliferate. Nutrientsand growth factors are added periodically as desired. To increase therate at which the number of cells 25 increase, the mesenchymal stemcells or fibroblast can be genetically altered to increase localproduction of desired growth factors. This can be done by viraltransfection or by incorporating plasmid genes into the matrix of thebiocompatible material.

[0038] The mesenchymal stem cells are directed to differentiate intofibroblasts and subsequently to fibrocytes. Differentiation occurs undersuitable conditions such as exposure to growth factors. Matrix materialsare then produced by the fibrocytes. A cyclic tensile load introducedthrough sheet 15 stimulates the fibrocytes to orientate and align in thedirection of tension. Fibrous tissue forms on sheet 15 over a period oftime and sheet 15 is then rolled up along strips 105. The ends 80 ofsheet 15 are crimped and then clamped in a cyclic loading machine asschematically depicted in FIG. 19. Further cyclic tensile load isapplied to the structure immersed in culture medium 110 to promotegrowth, organization and maturation of cells 25. Tissue-engineeredligament 10 is then implanted into a patient when there is sufficientmaturation.

EXAMPLE II

[0039] Another method for cell seeding involves clamping sheet 15 atboth ends 80 of the patterned sections 90, 95 and immersing sheet 15 inculture medium solution 110. Sheet 15 is initially held in tension andmesenchymal stem cells or fibroblasts are introduced into solution 110and onto strips 105. Cells 25 are allowed to attach to strips 105 andproliferate. Nutrients and growth factors are added periodically. Toincrease the rate at which the number of cells 25 increase, themesenchymal stem cells or fibroblast can be genetically altered toincrease local production of desired growth factors. This can be done byviral transfection or by incorporating plasmid genes into the matrix ofthe biocompatible material.

[0040] Once there is evidence of cell adhesion, sheet 15 is rolled-upalong the strips 105 to form a slender structure 35. Ends 80 are thencrimped and then clamped. As the rolled-up slender structure 35continues to be immersed in culture medium 110, a cyclic tensile load isapplied to structure 35. The mesenchymal stem cells now differentiateinto fibroblasts and secretion of matrix materials takes place. Thefibroblasts form fibrocytes and these fibrocytes become aligned in thedirection of tension. Over a period of time, the fibrocytes mature intofibrous tissue which forms over rolled-up slender structure 35.

EXAMPLE III

[0041] A third method involves forming a slender structure 35 by any ofthe methods previously described. Both ends 80 are crimped and clamped,and immersed in culture medium solution 110. The slender structure 35 isinitially held in slight tension and mesenchymal stem cells areintroduced into the solution and onto the slender structure 35. Thecells attach to strips 105 and proliferate. Nutrients and growth factorsare added periodically as desired. To increase the rate at which thenumber of cells 25 increase, the mesenchymal stem cells or fibroblastcan be genetically altered to increase local production of desiredgrowth factors. This can be done by viral transfection or byincorporating plasmid genes into the matrix of the biocompatiblematerial.

[0042] The mesenchymal stem cells are directed to differentiate intofibroblasts and subsequently to fibrocytes under suitable conditions,including exposure to growth factors. Matrix materials are then producedby the fibrocytes. A cyclic tensile load is introduced to slenderstructure 35. As the ends of the slender structure 35 move towards andaway from one another, the space between strips 105 opens and closes,thereby permitting cells and nutrients to flow between the strips, andthereby giving the cells the opportunity to attach to the interior ofthe structure. This loading also stimulates cells 25 further. Thefibrocytes orientate and align in the direction of tension. Over aperiod of time, the fibrocytes mature into fibrous tissue which formsover slender structure 35.

[0043] The in-vitro tissue-engineered ligament is ready for implantationonce ligamentous tissue has formed on slender structure 35. Crimped ends80 of in-vitro tissue-engineered ligament 10 are then inserted andsecured into bone tunnels 65. Preferably, tissue-engineered ligament 10is secured with interference screws 70. Alternatively, other methodswell known in the art may be used.

[0044] Initially, the material of scaffold sheet 15 of tissue-engineeredligament 10 supports loading across the joint. For scaffold sheet 15made of biodegradable material, as the scaffold material of theimplanted tissue-engineered ligament 10 degrades over time, the load isgradually transferred to the newly formed tissue. Eventually, whenscaffold sheet 15 is completely absorbed, the entire load is transferredto the newly formed tissue of ligament 10.

[0045] The preferred embodiments described above contain many exampleswhich are not limitations on the scope of the invention butillustrations of alternate embodiments. Many other variations arepossible within the scope of the invention, as those skilled in the artwill recognize from the following claims.

1. An apparatus for reconstruction of a previously torn ligament, saidapparatus comprising: a scaffold of biocompatible material, the scaffoldhaving at least one layer forming a scaffold sheet; means for seedingthe scaffold sheet with fibrocyte forming cells to form a seededscaffold; means for increasing the number of the fibrocyte forming cellsseeded on the scaffold to create a tissue-engineered scaffold; and meansfor forming a slender structure from the tissue-engineered scaffold, theslender structure being a tissue-engineered ligament suitable forimplantation into a patient for reconstruction of a ligament. 2-10.(Canceled)
 11. The apparatus of claim 45 wherein the means for attachingis an interference screw for fixation of the end of each ligament at theimplantation site. 12-17. (Canceled)
 18. The apparatus of claim 1wherein the fibrocyte forming cells are fibroblast cells. 19-23.(Canceled)
 24. A method of making a tissue-engineered ligament, saidmethod comprising: forming a scaffold sheet of biocompatible materialhaving at least one layer; seeding the scaffold sheet with fibrocyteforming cells to form a seeded scaffold of at least one sheet;increasing the number of the fibrocyte forming cells on the seededscaffold to create a tissue-engineered scaffold; and forming a slenderstructure from the tissue-engineered scaffold, the slender structurebeing a tissue-engineered ligament suitable for implantation into apatient for reconstruction of a ligament.
 25. The method of claim 24further comprising attaching each end of the tissue-engineered ligamentto implantation sites within a patient.
 26. The method of claim 24wherein the scaffold is a single sheet.
 27. The method of claim 24wherein the scaffold is slit at least once, the slit is made in thedirection of a first end of the scaffold to a second end of thescaffold, and the slit terminates prior to the first end and the secondend.
 28. The method of claim 24 wherein seeding the scaffold withfibrocyte forming cells includes placing the scaffold into a culturedmedium containing the fibrocyte forming cells.
 29. The method of claim24 wherein increasing the number of fibrocyte cells includes incubatingthe scaffold sheet after seeding.
 30. The method of claim 25 whereinincreasing the number of fibrocyte cells further includes incubating thetissue-engineered ligament after forming the slender structure.
 31. Themethod of claim 24 wherein increasing the number of fibrocyte cellsincludes incubating the tissue-engineered ligament after forming theslender structure.
 32. The method of claim 24 wherein forming a slenderstructure includes rolling the scaffold sheet to form a rolled tubularstructure.
 33. The method of claim 24 wherein forming a slenderstructure includes folding the scaffold sheet to form an accordionstructure.
 34. The method of claim 24 wherein forming a slenderstructure includes cutting a series of strips from the scaffold sheetand stacking the series of strips on top of one another to form theslender structure.
 35. The method of claim 25 wherein attaching each endof the ligament to implantation sites within a patient involves using aninterference screw for fixation.
 36. The method of claim 24 furthercomprising molding threads into each end of the tissue-engineeredligament for implantation.
 37. The method of claim 24 further comprisingproviding interlocking surfaces on the scaffold sheet for locking thescaffold sheet into the tissue-engineered ligament when the method stepof forming a slender structure is accomplished.
 38. The method of claim24 further comprising applying cyclic tensile loading of the seededscaffold prior to implantation to increase the strength of the scaffold.39. The method of claim 24 further comprising applying cyclic tensileloading of the slender structure prior to implantation to strengthen thetissue-engineered scaffold.
 40. The method of claim 24 furthercomprising forming spaces within the slender structure to allow thefibrocyte forming cells to grow therein.
 41. The method of claim 24wherein increasing the number of fibrocyte forming cells furthercomprises implanting the seeded scaffold in a penultimate section of apatient's body prior to implantation in a functional location.
 42. Themethod of claim 24 wherein increasing the number of fibrocyte formingcells further comprises implanting the slender structure in apenultimate location of a patient's body prior to implantation in afunctional location.
 43. The method of claim 24 further comprisingcoating the scaffold sheet with a material to promote adhesion of thefibrocyte forming cells.
 44. The method of claim 24 further comprisinggenetically altering fibrocyte forming cells to increase production offibrocyte cells.
 45. The apparatus of claim 1 wherein the apparatusfurther comprises: means for attaching each end of the tissue-engineeredligament to implantation sites within a patient.
 46. (Canceled)
 47. Themethod of claim 24 further comprising forming microchannels in thescaffold sheet.
 48. A tissue engineering ligament formed by forming ascaffold sheet, seeding the scaffold sheet with cells, increasing thenumber of cells, and forming a slender structure suitable for use as aligament.